Apparatus for synthesis of peptides or the like organic compounds

ABSTRACT

An apparatus for synthesis of peptides or the like organic compounds, in which transfer of the selected reagents into and out of a reaction vessel is conducted by utilizing vacuum and a pressurized inert gas under control of various valves. No liquid pumps are required. The sequential operation of the valves may be automatically controlled by a programmer.

United States Patent Kubodera et al.

[ Mar. 7, 1972 [54] APPARATUS FOR SYNTHESIS OF PEPTIDES OR THE LIKEORGANIC COMPOUNDS Toshiya Kubodera; Tasuku Hara; IIideki Makabe, all ofKyoto, Japan Shimadzu Seisakusho Ltd., Kyoto, Japan July 3, 1969 [72]Inventors:

Assignee:

Filed:

Appl. No.:

Foreign Application Priority Data July 8, 1968 Japan ..43/48048 U.S. Cl...23/252 R, 23/253 R, 23/259, 260/1 12.5 Int. Cl ..C07c 103/52 Field ofSearch ..23/252, 253, 254, 260, 285; 260/1 12.5

Edman et a1., A Protein Sequenator," European Journal of Biochemistry,1967, pp. 80- 91.

Primary Examiner-Morris O. Wolk Assistant Examiner-R. E. SerwinAttorney-Fidelman, Wolffe and Leitner [57] ABSTRACT An apparatus forsynthesis of peptides or the like organic compounds, in which transferof the selected reagents into and out of a reaction vessel is conductedby utilizing vacuum and a pressurized inert gas under control of variousvalves. No liquid pumps are required. The sequential operation of thevalves may be automatically controlled by a programmer.

7 Claims, 3 Drawing Figures PROGRAMMER APPARATUS FOR SYNTHESIS OFPEPTIDES OR THE LIKE ORGANIC COMPOUNDS This invention relates to anapparatus for synthesis of complicated organic compounds such aspeptides, proteins and the like. The invention is particularlyapplicable to what is commonly referred to in the art as solid-phasepeptide synthesis and will be described with particular reference tothis method of synthesis. However, it will be apparent as the inventionis described, that it is also applicable to the synthesis of a number ofproteins and the like compounds.

As is well known, according to the solid-phase method of synthesis,t-BOC-amino acids are successively applied to the matrix of adivinylbenzene resin to grow thereon as a long peptide chain. Thismethod has indeed contributed very much to the science and art ofpeptide synthesis. However, the process requires a large number of stepsand, consequently, time. For example, to add one amino acid to thegrowing peptide chain 1 l reagents must be successively introduced intothe reaction vessel in as many as 28 steps, and the operation requiresseveral hours. The amount of a reagent required for one step of theprocess is proportional to the amount of the resin serving as aninsoluble solid support in the reaction and the molar ratio of the aminoacid first bonded to the resin. For synthesis of an ordinary peptide, ifthe reaction is to proceed 100 percent, the amount of an amino acid tobe introduced into the reaction vessel is three to four times the amountof the amino acid required for the reaction. For example, the amount ofthe reagent used in one step of the process is no less than to 40 ml.Since most of the reagents required for the reaction of synthesis areexpensive, their loss in the transfer lines of the apparatus must bereduced to minimum. In this connection, mixing of difierent reagentsremaining in the transfer lines must also be avoided. In order toshorten the total time required for all steps of the process tocomplete, selection of reagents and transfer thereof into and out of thereaction vessel must be conducted as quickly as possible, and in thereaction vessel it is required that the reagent selected and transferredinto the vessel should stay in the space between the upper and lowerglass filters during the course of reaction so as to be in good contactwith the divinylbenzene resin provided in the space.

The primary object of the invention is therefore to provided a new andimproved apparatus for synthesis of peptides, proteins or the likeorganic compounds, which completely meets the above-mentionedrequirements.

The apparatus of the invention comprises a reaction vessel, a pluralityof bottles containing reagents, a sampling vessel communicatable withthe reaction vessel and a selected one of the bottles, a device forproducing vacuum and a source of pressurized inert gas. The vacuum orreduced pressure is applied to the sampling vessel so as to withdraw apredetermined quantity of the selected reagent into the sampling vesselto be temporarily stored therein, and then the pressurized gas isintroduced into the sampling vessel so as to push the reagent out of thesampling vessel into the reaction vessel. The selection and transfer ofthe reagents are performed by operating various valves included in theapparatus in a predetermined sequential manner, and the sequentialoperation of the valves can be automatically controlled by a suitableprogrammer."

Since vacuum and the pressure of inert gas are utilized to transfer thereagents, the time required for the transfer is greatly reduced incomparison of the prior art devices which employ liquid pumps for thesame purpose.

The invention will be more clearly understood by reading Referring toFIG. 1, there are shown a plurality of bottles T1, T2, Tn eachcontaining a reagent R1, R2, Rn

required for synthesis of a desired peptide. A selector valve A isprovided having a plurality of input ports A1, A2, An and a singleoutput port A0. The bottles Tl-Tn are connected to the input ports Al-Anthrough suitable pipes 10-1, 10-2, l0-n, respectively, while a samplingvessel 2 is connected to the output port Ao through a suitable pipe10-0. The vessel 2 is provided to temporarily store a predeterminedquantity of a reagent selected by the selector valve A from the bottlesin the manner to be described hereinafter. A valve B is provided tosequentially effect the transfer of the reagent from the selected bottleinto the vessel 2 and thence into a reaction vessel to be describedlater. The valve B has four ports B0, B1, B2 and B3. The port B1 isconnected to the sampling vessel 2 through a pipe 11; the port B2, to ametering chamber 4 through a pipe 12 in which an electromagnetic vacuumvalve E is inserted; the port B2, to a bomb 3 containing an inert gassuch as argon or nitrogen through a pipe 13 in which a gas regulator His inserted; and the port B3 is open to atmosphere. The chamber 4determines the quantity of a reagent to be withdrawn from the selectedone of the bottles Tl-Tn into the sampling vessel 2. The inner volume ofthe chamber 4 may have a fixed value provided that it is large enough toenable the introduction into the vessel 2 of a reagent in such an amountas is required for the reaction in the vessel. The chamber 4 may also bevariable in volume, or so designed that it can be automatically set toan optimum volume for each reagent. The chamber 4 is connected to avacuum pump 5 through a suitable pipe 14 in which a vacuum valve F andan electromagnetic three-way valve G having ports a, b and c areinserted. The valve G selectively connects the pump 5 to the chamber 4through the pipe 14 and to a waste vessel 7 through a pipe 15.

The sampling vessel 2 has an outlet opening 2', which is connectedthrough a pipe 16 to a unidirectional valve D such as a ball valve sodesigned as to pass the flow of reagents through the pipe 16 in thedirection of an arrow X but to block the flow therethrough in theopposite direction. A valve C is provided having three input ports C1,C2 and C3 and one output port Co. The port C1 is connected to the outputside of the unidirectional valve D through a pipe 17; the port C2, tothe bomb 3 through a pipe 18 in which a flow meter I is inserted; theport C3, to the waste vessel 7 through a pipe 19; and the output portCo, to a reaction vessel 6 through a pipe 20.

As shown in detail in FIG. 2, the reaction vessel 6 encloses therein alower glass filter 6A disposed adjacent the bottom of the vessel, alayer of divinylbenzene resin 6B disposed closely on the lower filterand an upper filter 6C far above the layer 68. The reagent is introducedinto the vessel 6 through an inlet opening 6 formed in the bottom of thevessel and passes through the lower filter 6A to stay in the spacebetween the lower and upper filters.

A programmer 8 controls and sequences the operations of the vacuumvalves A to G.

The sequential operation of the apparatus will now be described withparticular reference to the reagent R1 in the bottle Tl.

First, in the valve A the input port A1 is connected to the output portAo, so that the reagent R1 in the bottle T1 is ready to be withdrawn.Then, the valve E is closed and the valve F is opened, with the valve Gbeing so set that the ports a and b are connected while the port 0 isclosed. This causes the vacuum pump 5 to evacuate air from the meteringchamber 4. The valve B is then so operated that the ports Bo and B1communicate as shown in FIG. 1, and the valve F is closed and the valveE, opened. (Simultaneously with the closing of the valve F, the port aof the valve G may also be disconnected from the port b and connected tothe port c.) The opening of the valve E causes the pressure in thesampling vessel 2 to be reduced by a level corresponding to the volumeof the chamber 4, so that the reagent R1 is withdrawn from the bottle Tlinto the vessel 2. At this time, the valve D is closed due to the weightof the ball therein and the suction applied thereto from the chamber 4.The experiments conducted by the present inventors show that thequantity of the reagent that is introduced into the vessel 2 issubstantially proportional to the volume of the vessel 2 provided thatthe pressure in the chamber 4 is below 0.1 mm. Hg, which level isreached by evacuation of the chamber 4 for 10 to 15 seconds. FIG. 2 is agraph in which the amount in ml. of a reagent withdrawn into thesampling vessel 2 is plotted against the time in seconds of evacuationof the metering chamber 4. The curve A was resulted in case the volumeof the chamber 4 was 60 ml. and the curve B was resulted in case thevolume was 48 ml.

When the required quantity of the reagent has been introduced into thevessel 2, the valve E is closed, and the port B of the valve B isdisconnected from the port B1 and connected to the port B2, while theport Co of the valve C is connected to the port C1 as shown in H6. 1. Asa result, the inert gas in the bomb 3 is introduced through theregulator H and the valve B into the vessel 2 thereby to discharge thestored reagent out of the vessel 2. The reagent pushes theunidirectional valve D open to pass through the valve C into thereaction vessel 6 through the inlet opening 6 in the bottom thereof. Theregulator H is set so that the pressure of the inert gas is enough tocompletely push the reagent upwardly through the lower filter 6A so asto stay in the space between the upper and lower filters 6A and 6C. Evenwhen bubbling by the inert gas and shaking by a suitable mechanism, notshown, are conducted to accelerate the reaction in the vessel 6, thereagent can stay in the space between the upper and lower filters 6A and6C. This assures thorough contact between the reagent and the resin andprevents loss of the reagent. The pressure of the inert gas also helpspush the reagent remaining in the pipe -1 back into the bottle Tl.Therefore, no reagent remains in the transfer lines between the bottleT1 and the reaction vessel 2. This is a great advantage of the apparatusof the invention, since if any reagent used in one step of the processremained in the pipe, it would mix and react with the reagent to be usedin the next step thereby preventing the required reaction fromproceeding properly.

When the introduction of the reagent R1 into the reaction vessel 6 hasbeen completed, the port B0 of the valve B is connected to the port B3which is open to atmosphere and the port Co of the valve C is connectedto the port C2, whereupon the inert gas from the bomb 3 is introducedinto the reaction vessel 6, wherein the gas causes bubbling to advancethe reaction therein. After a predetermined period of time, the port Coof the valve C is connected to the port C3 and thence to the wastevessel 7 and the ports a and c of the three-way valve G are connected,so that the vacuum produced by the pump 5 is applied to the reactionvessel 6 through the valve G, the pipes l5, 19, the valve C and the pipe20. This causes any excess reagent remaining in the vessel 6 to bewithdrawn therefrom into the waste vessel 7. When the evacuation of thereaction vessel 6 has been completed, the valves G and C are restored tothe original conditions as shown in FIG. 1, thereby completing this onestep of the process.

With respect to the other reagents, the operation of the apparatus isthe same as that described just above so that no particular descriptionthereof will be required.

Thus, in accordance with the invention, all of the operations involvedin the synthesis of peptides can be performed by operating the variousvalves in a predetermined sequential manner, and the sequentialoperation of the valves can be automatically controlled by a programmer.Since vacuum and the pressure of an inert gas are utilized to transferthe reagents, the time required for the operation of the apparatus isgreatly reduced, and mixing of reagents used in different steps of theprocess can be completely avoided.

What we claim is:

I. In an apparatus for synthesis of organic compounds such as peptides,proteins, and the like, comprising a reaction vessel, a plurality ofreagent bottles each containing a reagent required for said synthesis asam ling vessel, a first means for selectively connecting said samp mgvessel to one of said plurality of reagent bottles and to said reactionvessel, and reagent transfer means, the improvement in said reagenttransfer means comprising a source of pressurized gas, a source ofreduced pressure, and a second means for selectively connecting saidsource of pressurized gas and said source of reduced pressure to saidsampling vessel, whereby transfer of reagent from one of said pluralityof reagent bottles to said sampling vessel is effected by connectingsaid source of reduced pressure to said sampling vessel and transfer ofreagent from said sampling vessel to said reaction vessel and excessreagent in said first means is returned to said one of said plurality ofreagent bottles are effected by connecting said source of pressurizedgas to said sampling vessel.

2. The apparatus of claim 1, wherein said reduced pressureproducingmeans comprises a chamber and a vacuum pump connected thereto forproducing a reduced pressure in said chamber.

3. The apparatus of claim 2, wherein said chamber is fixed in volume.

4. The apparatus of claim 2, wherein said chamber is variable in volume.

5. The apparatus of claim 1, wherein said second connecting meansconnects said reaction vessel to said source of pressurized gas aftersaid reagent is pushed into said reaction vessel, so that said gas isintroduced in said reaction vessel to cause bubbling therein.

6. The apparatus of claim 1, wherein said second connecting meansconnects said reduced pressure producing means to said reaction vesselafter the reaction therein for one step of the process of synthesis hasbeen completed, and further including a waste vessel interposed betweensaid reaction vessel and said reduced pressure-producing means so thatany reagent remaining in said reaction vessel after completion of thereaction for one step of the process may be withdrawn by said reducedpressure into said waste vessel.

7. The apparatus of claim 1, further including a programmer forcontrolling the operations of said first, second and third connectingmeans in accordance with a predetermined program.

2. The apparatus of claim 1, wherein said reduced pressure-producing means comprises a chamber and a vacuum pump connected thereto for producing a reduced pressure in said chamber.
 3. The apparatus of claim 2, wherein said chamber is fixed in volume.
 4. The apparatus of claim 2, wherein said chamber is variable in volume.
 5. The apparatus of claim 1, wherein said second connecting means connects said reaction vessel to said source of pressurized gas after said reagent is pushed into said reaction vessel, so that said gas is introduced in said reaction vessel to cause bubbling therein.
 6. The apparatus of claim 1, wherein said second connecting means connects said reduced pressure producing means to said reaction vessel after the reaction therein for one step of the process of synthesis has been completed, and further including a waste vessel interposed between said reaction vessel and said reduced pressure-producing means so that any reagent remaining in said reaction vessel after completion of the reactiOn for one step of the process may be withdrawn by said reduced pressure into said waste vessel.
 7. The apparatus of claim 1, further including a programmer for controlling the operations of said first, second and third connecting means in accordance with a predetermined program. 